Discovery of Diphenoxy Derivatives with Flexible Linkers as Ligands for β-Amyloid Plaques

Article abstract of DOI:10.1021/acs.molpharmaceut.0c00537

The highly rigid and planar scaffolds with π-conjugated systems have been widely considered to be indispensable for β-amyloid (Aβ) binding ligands. In this study, a library of diphenoxy compounds with different types of more flexible linkers as Aβ ligands were synthesized and evaluated. Most of them displayed good affinity (K_i< 100 nM) for Aβ_1-42aggregates, and some ligands even showed values of K_iless than 10 nM. Structure-activity relationship analysis revealed that modification on the linkers or substituents tolerated great flexibility, which challenged the long-held belief that rigid and planar structures are exclusively favored for Aβ binding. Three ligands were labeled by iodine-125, and they exhibited good properties in vitro and in vivo, which further supported that this flexible scaffold was potential and promising for the development of Aβ imaging agents.

Full text of DOI:10.1021/acs.molpharmaceut.0c00537

Subscriber access provided by University of Rochester | River Campus &amp; Miner Libraries
Article
Discovery of Diphenoxy Derivatives with Flexible
Linkers as Ligands for #-amyloid Plaques
Jianhua Jia, Longfei Zhang, Jia Song, Jiapei Dai, and Mengchao Cui
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/
acs.molpharmaceut.0c00537 • Publication Date (Web): 26 Aug 2020
Downloaded from pubs.acs.org on August 26, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 43
Molecular Pharmaceutics
1
2
3
4
5
Discovery of Diphenoxy Derivatives with Flexible Linkers as Ligands for
6
7
8
β-amyloid Plaques
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Jianhua Jia†, ‡, Longfei Zhang†, Jia Song†, Jiapei Dai§, Mengchao Cui*,†
† Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing
Normal University, Beijing 100875, P. R. China.
‡ Department of Radiological Medicine and Oncology, College of Basic Medicine, Chongqing
Medical University, Chongqing 400016, P. R. China.
§ Wuhan Institute for Neuroscience and Neuroengineering, South-Central University for
Nationalities, Wuhan 430074, P. R. China.
1
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 2 of 43
1
2
3
4
5
ABSTRACT
6
7
8
9
The highly rigid and planar scaffolds with π-conjugated systems have been widely considered to be
indispensable for Aβ binding ligands. In this study, a library of diphenoxy compounds with different
types of more flexible linkers as Aβ ligands were synthesized and evaluated. Most of them displayed
good affinity (Ki < 100 nM) for Aβ1-42 aggregates, and some ligands even showed the values of Ki
being less than 10 nM. Structure-activity relationship analysis revealed that modification on the
linkers or substituents tolerated great flexibility, which challenged the long-held belief that rigid and
planar structure are exclusively favored for Aβ binding. Three ligands were labeled by iodine-125,
and they exhibited good properties in vitro and in vivo, which further supported that this flexible
scaffold was potential and promising for the development of Aβ imaging agents.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
KEYWORDS: Diphenoxy derivatives, Flexible linkers, β-amyloid, Structure-Activity Relationship
2
ACS Paragon Plus Environment

Page 3 of 43
Molecular Pharmaceutics
1
2
3
4
5
INTRODUCTION
6
7
8
9
Alzheimer's disease (AD), a common dementia in the elderly, brings a huge and disastrous threat to
the old, their families, and society, made worse with the global population aging trend. To date, the
effectiveness of therapeutic medicine is far from satisfying, and the diagnosis of the disease still
faces many difficulties in the clinic, even as the research on its pathogenesis and pathological
development continues.1-3 According to “amyloid hypothesis”, amyloid-beta (Aβ) peptides in the
brain play a significant role in AD onset and progression.4 In the last twenty years, based on this
hypothesis, developing molecular probes targeting Aβ for positron emission tomography (PET) or
single-photon emission computed tomography (SPECT) imaging, aiming for early diagnosis of AD,
is a hot research topic.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Three broad categories of Aβ ligands are currently under investigation (Figure 1). One class is
derived from the dye Congo red (CR), including the direct CR analogs (Chrysamine G5, X-346,
Me-X047, IMSB8, etc.) and the analogs with partial structure of CR (the stilbenes and DDNPs).
Among them, two imaging agents with the styrylbenzene backbone, [18F]AV-45 (florbetapir)9 and
[18F]BAY94-9172 (florbetaben)10 have been recently approved by U.S. FDA successively, besides,
[18F]FDDNP11 was the first 18F-labelled PET probe for evaluating in the brain of living AD patients
since it is capable of binding to two hallmarks of AD pathology, namely, tangles and plaques. A
second class is derived from the dye thioflavin-T (Th-T), including benzothiazoles12-16
,
benzoxazoles17-19, benzofurans20-22, benzoimidazoles23, imidazopyridines24-26, benzothiophenes27, etc.
Among them, [11C]PIB15 has been used to conduct more than 10000 PET scans worldwide, and its
analog [18F]GE-067 (flutemetamol)16 was approved by U.S. FDA, moreover, a very promising
SPECT imaging agent [123I]IMPY28 was declared a failure after human studies, but it's analog
3
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 4 of 43
1
2
3
4
5
6
7
8
9
[123I]DRM-10629 was currently under clinical studies. The final class was the plant pigments and
their derivatives, including curcumin30, dibenzylideneacetone31, flavone32, styrylchromone33,
chalcone34, aurone35, diphenylpropynone36, etc. After composite analysis the structure features of
these probes, we can conclude that all of them have similar conjugated aromatic structure, which
presents a rigid and relatively planar configuration, and this common feature has been widely
considered to be indispensable for Aβ binding.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CH3
N
NH2
H2N
N
N
N
N
N
S
H3C
HO
Cong Red (CR)
Thioflavin T (ThT)
NaO3S
SO3Na
R2
NH
N
18F
NH
S
O
O
X
R1
R1 = H, R2 = 11CH3
18F]GE-067:
O
[
[
11C]PIB:
[
18F]AV-45:
X = N
18F]BAY94-9172:
R1 = 18F, R2 = CH3
[
X = CH
18F
N
R
CN
N
123I
NC
N
[
123I]IMPY:
R = NMe2
N
NH
18F]FDDNP
[
123I]DRM-106:
R =
[
Figure 1. Classic dyes for Aβ plaques and derived PET/SPECT imaging agents.
Recently, our group made an “out of box” innovation to design and evaluate a series of
benzyloxybenzene derivatives as flexible Aβ binding probes. This flexible scaffold with a rotatable
benzyloxy linker shared the same binding site as [125I]IMPY, a rigid and planer ligand37. The
research hinted the binding pocket of Aβ fiber had considerable tolerance to a certain degree of
flexible ligands. Besides, Th-T and its analogs were reported to display a complex binding mode on
Aβ fibrils38, 39. To investigate the binding of flexible ligands to Aβ fibrils, in this study, a series of
diphenoxy derivatives with different types of flexible linkers (alkane, polyethylene glycol (PEG),
4
ACS Paragon Plus Environment

Page 5 of 43
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
2-substituted propane, and di-substituted piperazine) were designed, synthesized and evaluated
(Figure 2). Meanwhile, we investigated the possible structure-activity relationship (SAR) of these
ligands for Aβ aggregates, believing the SAR results would afford a more rational design of novel
flexible ligands and give a new sight of understanding the binding mode between these ligands and
Aβ fibril. In addition, to further confirm the applicability of the flexible ligands, three radioiodinated
diphenoxy derivatives with high affinity were screened and evaluated as potential SPECT probes for
the detection of Aβ plaques in AD brains.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
flexible linker
R1
N
O
O
n
R2
n = 1 - 11
O
O
I
n
n = 2, 3
Y
R
O
O
R
Y = OH, F
nO
n = 2, 6
N
N
O
I
N
n
Figure 2. The structure of diphenoxy derivatives with flexible linkage as novel Aβ binding probes.
MATERIALS AND METHODS
General Information. All reagents used in the synthesis were commercial products and were used
without further purification. No-carrier-added [125I]NaI (nominal molar activity 2200 Ci/mmol) was
1
13
purchased from Perkin-Elmer. The H and C-NMR was obtained at 400 and 100 MHz on Bruker
Avance III NMR spectrometer in CDCl3 at room temperature with TMS as internal standard. High
5
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 6 of 43
1
2
3
4
5
6
7
8
9
resolution mass spectra (HRMS) were acquired on a Thermo scientific Q-Exactive (ESI) (USA)
mass spectrometer. Reactions were monitored by TLC (silica gel 60 F254 aluminum sheets, Merck),
and compounds were visualized by illumination with UV lamp (λ = 254 nm). Column
chromatography purification was performed on silica gel (54 - 74 μm) from Qingdao Haiyang
Chemical Co., Ltd. Radiochemical purity was determined by HPLC performed on a Shimadzu
SCL-20 AVP system equipped with a SPD-20A UV detector (λ = 254 nm) and Bioscan Flow
Count 3200 NaI/PMT γ-radiation scintillation detector. HPLC separations and analysis were achieved on a
Venusil MP C18 reverse phase column (Bonna-Agela Technologies, 5 µm, 4.6 mm × 250 mm)
eluted with an isocratic system at a flow rate of 1.0 mL/min. Mobile phase A was water and mobile
phase B was acetonitrile. Fluorescent images were obtained using an Axio Observer Z1 (Zeiss,
Germany). Tg mice (C57BL6, APPswe/PSEN1, 11 months old, female), used as an AD model, and
wild-type control (C57BL6, 11 months old, female) were purchased from Zhishan (Beijing) Institute
Of Healthcare Research Co., Ltd., and the Institute of Laboratory Animal Science, Chinese Academy
of Medical Sciences. Normal mice (ICR, 5 weeks, male) used for the biodistribution experiments
were purchased from Vital River Laboratories. All protocols requiring the use of animals were
approved by the animal care committee of Beijing Normal University. Post-mortem brain tissues
from autopsy-confirmed AD patients (91 years old, male, temporal lobe; 64 years old, female,
temporal lobe) were obtained from the Chinese Brain Bank Center.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis. General procedures for the synthesis of 1 - 12, 105, 107 and 108. To a solution of
4-nitrophenol (1.0 equiv.) or 4-iodophenol (1.0 equiv.) and 1, n-dibromoalkane (n = 2 – 8, 10, 12; 2.0
equiv.) in anhydrous DMF (10 mL) was added K2CO3 (1.5 equiv.) as base. The resulting mixture
6
ACS Paragon Plus Environment

Page 7 of 43
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
was stirred vigorously at 90 °C for 2 h, and DMF was removed in vacuum at 110 °C. After cooling
to room temperature, 30 mL of water was added and extracted by dichloromethane (3 × 20 mL).
Collected organic layers were dried over anhydrous MgSO4, filtered, and concentrated under vacuum.
The crude mixture was purified by recrystallization or silica gel column chromatography.
General procedures for the synthesis of 13 - 50, 130 and 131. To a solution of substituted phenols
(1.0 equiv.) and bromine compounds (1.0 equiv.) in anhydrous DMF (10 mL) was added K2CO3 (1.5
equiv.). The similar post processing method was employed to afford compounds 13 - 50, 130 and
131 as the preparation of 1 - 12.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
General procedure for the synthesis of 51 - 53, 55, 58 - 60, 62 - 68, 71 - 76, 113, 114, 150 – 152. To
.
a solution of corresponding nitro-compounds (1.0 equiv.) and SnCl2 2H2O (4.0 equiv.) in EtOH (20
mL) was added concentrated HCl (5 mL). The resulting mixture was stirred at 110 °C for 4 h. After
EtOH was removed in vacuum, ammonia hydroxide was added to the mixture until pH > 7 in ice
bath. The mixture was extracted by CH2Cl2 (3 × 30 mL). Collected organic layers were dried over
MgSO4, filtered, and concentrated under vacuum. The crude mixture was purified by
recrystallization or silica gel column chromatography.
General procedure for the synthesis of 54, 56, 57, 61, 69, 70, 119, 137 and 138. To a solution of
corresponding nitro-compounds (1.0 equiv.) and hydrazine hydrate (5.0 equiv.) in EtOH (20 mL)
was added palladium/C catalyst (0.1 equiv.). The resulting mixture was stirred at 110 °C for 2 h, and
then filtered immediately; the filtrate was concentrated under vacuum. The products could be
obtained with high purity without further purification.
General procedure for the synthesis of 77 – 102, 106, 115, 116, 120, 141 – 144 and 153 - 155. To a
solution of amino compound (1.0 equiv.) and paraformaldehyde (10.0 equiv.) in acetic acid (20 mL)
7
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 8 of 43
1
2
3
4
5
6
7
8
9
in ice bath was added NaBH3CN (5.0 equiv.) in batches. The mixture was stirred vigorously at room
temperature for 12 h. Then water (10 mL) and ammonia hydroxide was added to the mixture until pH
> 7 in ice bath. The resulting mixture was extracted by CH2Cl2 (3 × 40 mL). The organic layers were
dried over MgSO4, filtered, and concentrated under vacuum. The crude product was purified by
recrystallization or silica gel column chromatography.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
General procedure for the synthesis of 103, 104 and 117. To a solution of 77 (or 90, or 115, 1.0
equiv.) and (Ph3P)4Pd (0.1 equiv.) in toluene (10 mL) and Et3N (3 mL) was added (Bu3Sn)2 (3.0
equiv.). The mixture was stirred under reflux overnight. The solvent was evaporated under vacuum
and the crude product was purified by silica gel chromatography.
General procedure for the synthesis of 109 and 110. To a solution of 107 (or 108, 1.0 equiv.) and
tosyl chloride (1.2 equiv.) in CH2Cl2 (20 mL) was added Et3N (5 mL). The mixture was stirred at
room temperature for 4 h. Then 30 mL of water was added to the mixture and it was extracted by
CH2Cl2 (3 × 20 mL). The organic layer was collected, dried over MgSO4, and concentrated. The
residue was further purified by silica gel chromatography.
General procedure for the synthesis of 111 and 112. To a solution of 109 (or 110, 1.0 equiv.) and
4-iodophenol (1.0 equiv.) in acetone (20 mL) was added K2CO3 (1.5 equiv.) as base and 18-crown-6
(0.1 equiv.) as catalyst. The reaction mixture was stirred at 70 °C for 4 h, then the solvent was
removed under vacuum and 30 mL of water was added. The mixture was extracted with CH2Cl2 (3 ×
30 mL), then the organic layers were combined, dried by MgSO4 and concentrated in vacuum to give
a residue, which was further purified by silica gel chromatography.
The procedure for the synthesis of 118. A solution of 4-nitrophenol (3.82 g, 27.5 mmol) and NaOH
(1.10 g, 27.5 mmol) in water (25 mL) was heated until the solid was completely dissolved, and then
8
ACS Paragon Plus Environment

Page 9 of 43
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
2-(chloromethyl)oxirane (1.10 g, 13 mmol) was added slowly to the mixture. It was kept heating at
110 °C for 3 h. Yellow precipitate was generated, filtered, washed with EtOH (2 mL) and water (10
mL) to afford 118 without further purification.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The procedure for the synthesis of 121. To a solution of 120 (715.0 mg, 2.6 mmol) and
paraformaldehyde (313.1 mg, 20.5 mmol) in methanol (20 mL) was added NaOCH3 (5.2 mL, 1
mol/L in methanol). The resulting mixture was stirred intensely under reflux for 2 h, then NaBH4
(786.2 mg, 20.5 mmol) was added to the solution after cooling to room temperature. The mixture
was continued to stir under reflux for 2 h. The solvent was removed under vacuum, and 30 mL of
water was added and the mixture was extracted using CH2Cl2 (3 × 30 mL), then the combined
organic layers were dried by anhydrous MgSO4, filtered and concentrated to give crude product,
which was further purified by silica gel chromatography.
The procedure for the synthesis of 122. To a solution of 121 (782.1 mg, 2.6 mmol) and
tertbutyldimethylsilyl chloride (2.35 g, 15.6 mmol) in CH2Cl2 (20 mL) was added imidazole (1.06 g,
15.6 mg). The mixture was stirred at 40 °C for 3 h, and then filtered to remove the residue. The
filtrate was evaporated at vacuum and purified by silica gel chromatography.
The procedure for the synthesis of 123. To a solution of 122 (1.04 g, 2.5 mmol) in THF (15 mL) was
added di-tert-butyl dicarbonate (2.73 g, 12.5 mmol). The mixture was stirred at 85 °C for 4 h, and
then the solvent was removed by vacuum and purified by silica gel chromatography.
The procedure for the synthesis of 124. To a solution of 123 (761.3 mg, 1.2 mmol) in THF (15 mL)
was added tetrabutylammonium fluoride (6.0 mmol, 1 M in THF). The resulting mixture was stirred
at 30 °C for 5 min, and then the solvent was removed by vacuum and purified by silica gel
chromatography.
9
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 10 of 43
1
2
3
4
5
6
7
8
9
The procedure for the synthesis of 125. To a solution of 124 (164.3 mg, 0.3 mmol) in anhydrous
CH2Cl2 (10 mL) was added diethylaminosulfurtrifluoride (473.3 mg, 1.5 mmol) slowly at -78 °C.
The mixture was stirred from -78 °C to room temperature for 4 h, and then the reaction was
terminated by adding NaHSO3 saturated solution (10 mL). The mixture was washed by NaHCO3
saturated solution (3 × 20 mL) and extracted by CH2Cl2 (3 × 20 mL). The collected organic layers
were dried by anhydrous MgSO4, filtered and concentrated to give crude product, which was further
purified by silica gel chromatography.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
General procedure for the synthesis of 126, 145 and 146. To a solution of 125 (or 143, or 144, 0.2
mmol) in CH2Cl2 (10 mL) was added trifluoroacetic acid (1 mL). The resulting mixture was stirred at
room temperature for 1 h and evaporated at vacuum to afford a residue, and purified by silica gel
chromatography.
General procedure for the synthesis of 132 - 134. A solution of piperazine (1.0 equiv.), hydrobromic
acid (a.q. 40%, 1.0 equiv.) and compound 1, 4 or 7 (0.5 equiv.) in ethanol (20 mL) was reflux for 48
h, after reaction, the solvent was removed under vacuum. Hydrochloric acid (10%) and water was
added to the residue to adjust pH value of 6. The mixture was extracted with CH2Cl2 (3 × 30 mL),
and the water layers were collected and alkalized using NaOH (1 M) until pH > 9. The solution was
extracted again with CH2Cl2 (3 × 30 mL), the collected organic layer was dried by MgSO4 and
concentrated in vacuum to give the products 132 - 134.
General procedure for the synthesis of 139 and 140. A solution of 133 or 134 (1.0 equiv.) and
di-tert-butyl dicarbonate (5.0 equiv.) in THF (15 mL) was stirred at 85 °C for 4 h. After reaction, the
crude product 135 or 136 was extracted and then reduced by hydrazine hydrate/palladium-carbon
system to afford 139 or 140 .
10
ACS Paragon Plus Environment

Page 11 of 43
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
General procedure for the synthesis of 147, 148 and 149. To a solution of 132 (or 133, or 134, 1.0
equiv.) and 1-(2-bromoethoxy)-4-iodobenzene (2, 1.0 equiv.) in DMF (10 mL) was added Cs2CO3
(1.5 equiv.) as base. The reaction mixture was stirred at 90 °C for 4 h, and then DMF was evaporated
in vacuum at 110 °C. Water (30 mL) was added to the residue and the mixture was extracted using
CH2Cl2 (3 × 30 mL), then the combined organic layers were dried by anhydrous MgSO4, filtered and
concentrated to give crude product, which was further purified by silica gel chromatography.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Radiolabeling. The radio-iodinated ligands [125I]77, [125I]90 and [125I]115 were prepared by
iododestannylation reaction according to the methods reported previously.40 The final products were
purified by RP-HPLC. The separation conditions: Venusil MP C18 column (Bonna-Agela
Technologies, 4.6 mm × 250 mm), 1 mL/min, UV = 254 nm, CH3CN/H2O = 65/35 for [125I]77,
CH3CN/H2O = 70/30 for [125I]90, CH3CN/H2O = 85/15 for [125I]115. The retention time of the
radio-iodinated ligands and the corresponding cold iodinated compounds were compared by
co-injection and co-elution on RP-HPLC. The analysis conditions: Venusil MP C18 column
(Bonna-Agela Technologies, 4.6 mm × 250 mm), 1 mL/min, UV = 254 nm, CH3CN/H2O = 90/10.
Partition coefficient determination. The partition coefficients of radio-iodinated ligands were
measured according to a previously reported method.19
Biological evaluations. Biological evaluations including in vitro binding assay using Aβ1-42
aggregation, in vitro autoradiography using brain sections from Tg mice, wild-type mice and AD
patients, biodistribution studies using ICR normal mice were all conducted as previously reported
11
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 12 of 43
1
2
3
4
5
methods.19
6
7
8
9
RESULTS
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis. The diphenoxy derivatives bearing different lengths of alkane linkage and various
substituents were synthesized via two successive nucleophilic reactions (Scheme 1). Briefly, 1,
n-dibromoalkane (n = 2 – 8, 10, 12) was conjugated with the para-substituted phenols to give
bromine compounds 1 – 12 (24.3 – 62.1%). The diphenoxy derivatives 13 - 50 were obtained
through bromine compounds conjugating to various substituted phenols. The dimethylamino
derivatives 77 - 102, were prepared through reduction of the nitro compounds 13 - 21, 28 - 40 and 47
- 50 to produce amino compounds 51 - 76 using SnCl2/HCl or hydrazine hydrate/palladium
(C) reduction system, followed by reductive amination with paraformaldehyde and NaBH3CN in
acetic acid. Notably, due to the steric hindrance of o-NMe2 substituent for the compounds with short
alkane linkage, compounds 84 was obtained as a mono-methylated version. Besides, the
monophenoxy alkane derivatives 105 and 106 were obtained by similar methods (Scheme 2).
Scheme 1. Synthesis of diphenoxy ligands bearing different length of alkane linkage and various
substituents. a
R2
a
O
R1
OH
R1
O
n O
Br
n
13 - 104
1 - 12
R1
13 - 21
28 - 40
47 - 50
b
c
d
e
13 - 50
51 - 76
77 - 102
1 - 12
77 90
,
103 104
,
Compd
R1
R2
n
1
1
2
3
Compd
53
R1
R2
n
1
1
1
1
1
2
3
4
NO2
I
-
-
-
-
NH2
NH2
NH2
NH2
m-I
54
p-F
NO2
NO2
55
p-Cl
p-Br
56
12
ACS Paragon Plus Environment

Page 13 of 43
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
5
6
I
-
-
3
4
5
5
6
7
9
11
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
3
4
5
5
5
5
5
5
5
5
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
NH2
I
p-NH2
o-NH2
m-NH2
p-I
1
1
1
2
2
3
4
5
5
5
5
5
5
5
5
5
6
7
9
11
1
1
1
1
1
1
1
1
1
2
2
3
4
5
5
NO2
NO2
I
7
-
I
8
-
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
I
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
9
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
I
-
p-NH2
p-I
10
11
-
-
p-I
12
13
-
p-I
p-I
o-I
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
o-I
m-I
m-I
p-F
p-F
p-Cl
p-Br
p-NH2
o-NH2
m-NH2
p-I
p-Cl
p-Br
p-NO2
o-NO2
m-NO2
p-F
I
I
NH2
NH2
NH2
NH2
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
I
I
p-I
I
p-Cl
p-Br
p-I
p-I
I
p-I
I
p-I
I
p-OCH3
p-t-Bu3
p-I
o-I
I
m-I
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
I
p-F
p-NO2
p-I
p-Cl
p-Br
p- NMe2
o-NHMe
m-NMe2
p-I
p-I
p-I
o-I
I
m-I
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
p-F
p- NMe2
p-I
p-Cl
p-Br
p- NO2
o-NO2
p-I
p-I
o-I
13
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 14 of 43
1
2
3
4
5
6
7
8
9
40
41
42
43
44
45
46
47
48
49
50
51
52
I
m-NO2
p-F
5
5
92
93
NMe2
NMe2
NMe2
NMe2
NMe2
I
m-I
p-F
5
5
I
I
p-Cl
p-Br
p-I
5
94
p-Cl
5
I
5
95
p-Br
5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
I
5
96
p-NMe2
o-NMe2
m-NMe2
p-I
5
I
p-OCH3
p-t-Bu3
p-I
5
97
5
I
5
98
I
5
NO2
NO2
NO2
NO2
NH2
NH2
6
99
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
6
p-I
7
100
101
102
103
104
p-I
7
p-I
9
p-I
9
p-I
11
1
p-I
11
1
p-I
p-SnBu3
p-SnBu3
o-I
1
5
a Reagents and conditions: (a) 1, n-dibromoalkane, K2CO3, DMF, 90 °C, 2 h; (b) R2-phenol, K2CO3,
.
.
DMF, 90 °C, 2 h; (c) SnCl2 2H2O, HCl, EtOH, 110 °C, 4 h or N2H4 H2O, Pd/C, EtOH, 110 °C, 2 h;
(d) (HCHO)n, NaBH3CN, AcOH, 0 °C - r.t., 12 h; (e) (Ph3P)4Pd, (Bu3Sn)2, Et3N, toluene, 110 °C,
overnight.
Scheme 2. Synthesis of mono-phenoxy derivatives. a
OH
O
O
OH
a
b
I
I
N
O2N
106
105
a
Reagents and conditions: (a) 1-chlorohexane, K2CO3, DMF, 90 °C, 2 h; (b) 1. 1-chlorohexane,
.
K2CO3, DMF, 90 °C, 2 h; 2. SnCl2 2H2O, HCl, EtOH, 110 °C, 4 h; 3. (HCHO)n, NaBH3CN, AcOH,
0 °C - r.t., 12 h.
As displayed in Scheme 3, the diphenoxy derivatives bearing PEG linkage were prepared using a
similar method as above with some modifications. The bromide leaving group was replaced by the
tosylate group to enhance reactivity. The nitro compounds 111 and 112 were obtained in high yields
(89% and 98%, respectively), and the final dimethylamino compounds 115 and 116 were prepared
by the same methods described above.
14
ACS Paragon Plus Environment

Page 15 of 43
Molecular Pharmaceutics
1
2
3
4
5
6
Scheme 3. Synthesis of diphenoxy ligands bearing PEG linkage. a
7
8
9
I
O
OH
O
OH
OTs
c
n
b
a
n
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
n
O2N
O2N
O2N
107
108
n = 2;
n = 3;
111
112
n = 2;
n = 3;
109
110
n = 2;
n = 3;
O2N
I
SnBu3
I
e
f
O
O
O
d
O
O
2
O
n
n
N
N
H2N
113
114
115
116
n = 2;
n = 3;
n = 2;
n = 3;
117
a
Reagents
and
conditions:
(a)
2-(2-chloroethoxy)ethan-1-ol
or
2-(2-(2-chloroethoxy)ethoxy)ethan-1-ol, K2CO3, DMF, 90 °C, 2 h; (b) tosyl chloride, Et3N, CH2Cl2,
.
r.t., 4 h; (c) 4-iodophenol, K2CO3, 18-crown-6, acetone, 70 °C, 4 h; (d) SnCl2 2H2O, HCl, EtOH, 110
°C, 4 h; (e) (HCHO)n, NaBH3CN, AcOH, 0 °C - r.t., 12 h; (f) (Ph3P)4Pd, (Bu3Sn)2, Et3N, toluene,
110 °C, overnight.
The symmetric diphenoxy derivatives with 2-substituted propyl linkage were synthesized via the
methods shown in Scheme 4. The backbone compound 118 was obtained by a ring-opening and
substitute reaction starting from 2-(chloromethyl)oxirane and 4-nitrophenol in sodium hydroxide
aqueous solution. After reduction and methylation, the mono- and dimethylamino derivatives (121
and 120) were obtained successfully. Then the hydroxyl group of 120 was directly converted to
fluoride 127 using diethylaminosulfur trifluoride (DAST) in anhydrous CH2Cl2. For the
monomethylated compound 126, the hydroxyl and monomethylamino groups of 121 were protected
by tertbutyldimethylsilyl chlide (TBDMSCl) and ditertbutyl dicarbonate ((Boc)2O) successively to
give compound 122 and 123. After removing the TBS-protecting group of 123 with
tetrabutylammonium fluoride (TBAF), selective fluorination of 124 was performed by DAST to give
125, and the Boc-protecting group was removed using trifluoroacetic acid (TFA) to produce the final
monomethylated compound 126.
15
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 16 of 43
1
2
3
4
5
6
7
Scheme 4. Synthesis of fluorinated symmetric diphenoxy ligands. a
8
9
O
OH
Cl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
R1
O
O
R1
O2N
OH
a
118
R1 = NO2
R2
b
R3
N
R3
N
119
O
O
R1 = NH2
e
d
122
R2 = OTBS, R3 = H
c
121
120
R1 = NHMe
f
123
124
R2 = OTBS, R3 = Boc
R1 = NMe2
h
g
127
126
R2 = OH, R3 = Boc
R2 = F, R3 = Me
h
i
125
R2 = F, R3 = Boc
R2 = F, R3 = H
a
.
Reagents and conditions: (a) 2-(chloromethyl)oxirane, NaOH, H2O, 110 °C, 3 h; (b) N2H4 H2O,
Pd/C, EtOH, 110 °C, 2 h; (c) (HCHO)n, NaBH3CN, AcOH, 0 °C - r.t., 12 h; (d) 1. (HCHO)n,
NaOCH3, 90 °C, 2 h; 2. NaBH4, 90 °C, 2 h; (e) TBDMSCl, imidazole, CH2Cl2, 40 °C, 3 h; (f)
(Boc)2O, THF, 85 °C, 4 h; (g) TBAF (1M in THF), 30 °C, 5 min; (h) DAST, anhydrous CH2Cl2, -78
°C - r.t., 4 h; (i) TFA, CH2Cl2, r.t., 1 h.
The ligands bearing piperazine modified linkage were synthesized as shown in Scheme 5. Compound
129 with a methylene bridge between piperazine and two benzene ring was prepared by
aldehyde-ammonia condensation between intermediate 128 and 4-(dimethylamino)benzaldehyde in
CH2Cl2. The diphenoxy ligands with different lengths of alkane bridges between piperazine and
benzene ring (153, 154 and 155) were prepared according to the following methods. The bromine
compounds 1, 4 and 7 were conjugated to piperazine to give intermediate 132, 133 and 134, then the
intermediates were reacted separately with 1-(n-bromoethoxy)-4-iodobenzene (2, 5 and 8) to produce
the nitro diphenoxy derivatives 147, 148 and 149 using Cs2CO3 as base. The dimethylamino
derivatives 153, 154 and 155 were obtained according to the same procedures described above from
the nitro compounds. The mono-phenoxy derivatives (141, 142, 145 and 146) containing piperazine
16
ACS Paragon Plus Environment

Page 17 of 43
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
or piperidine moiety were prepared from the bromine compounds (4 and 7) and piperazine or
piperidine. The final products were obtained by reduction and methylation of compounds 130, 131,
135 and 136. For the piperazine compounds 145 and 146, the ring nitrogen atom needs protection
with tert-butyloxycarbonyl (Boc) and deprotection in the end.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 5. Synthesis of diphenoxy ligands bearing piperazine modified linkage and mono-phenoxy
derivatives containing piperazine or piperidine. a
N
N
Br
N
a
b
N
NH
I
I
I
129
128
137
138
n = 4, R = NH2;
n = 6, R = NH2;
d
d
c
Br
n
O
R
O2N
O
nN
e
1
n = 1;
4 n = 3;
130
131
n = 4, R = NO2;
n = 6, R = NO2;
141
142
n = 4, R = NMe2;
n = 6, R = NMe2;
7
n = 5;
f
Boc
NH
139
140
n = 4, R = NH2;
n = 6, R = NH2;
e
N
O
N
n
g
O
N
n
O2N
132
133
134
n = 2;
n = 4;
n = 6;
R
143
144
n = 4, R = NMe2;
n = 6, R = NMe2;
135
136
I
n = 4, R = NO2;
n = 6, R = NO2;
h
i
145
146
n = 4;
n = 6;
O
N
N
O
n
NH
nN
O
N
n
153
154
155
150
151
152
n = 2, R = NMe2;
n = 4, R = NMe2;
n = 6, R = NMe2.
147
148
149
n = 2, R = NH2;
n = 4, R = NH2;
n = 6, R = NH2;
n = 2, R = NO2;
n = 4, R = NO2;
n = 6, R = NO2;
R
j
e
a
Reagents and conditions: (a) piperazine, THF, 70 °C, 6 h; (b) 4-(dimethylamino)benzaldehyde,
.
NaBH(OAc)3, CH2Cl2, r.t., overnight; (c) piperidine, K2CO3, DMF, 90 °C, 2 h; (d) N2H4 H2O, Pd/C,
EtOH, 110 °C, 2 h; (e) (HCHO)n, NaBH3CN, AcOH, 0 °C - r.t., 12 h; (f) piperazine, HBr (a.q. 40%),
ethanol, 48 h; (g) (Boc)2O, THF, 85 °C, 3 h; (h) trifluoroacetic acid, CH2Cl2, 40 °C, 2 h. (i)
1-(n-bromoethoxy)-4-iodobenzene (2, 5 and 8; n = 2, 4 and 6), CsCO3, DMF, 90 °C, 4 h; (j)
.
SnCl2 2H2O, HCl, EtOH, 110 °C, 4 h.
In this study, 53 of target ligands were successfully synthesized and structurally validated. The
17
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 18 of 43
1
2
3
4
5
6
7
purities of these ligands were proved to be higher than 95% by high-performance liquid
chromatography (HPLC) (Table S1).
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Structure - activity relationships. The affinity of these flexible diphenoxy ligands for Aβ1-42
aggregates was measured by competition binding assays using [125I]IMPY as the competing
radioligand, which binds to the Th-T site on the surface of Aβ aggregates. IMPY was tested under
the same assay system for validation and comparison. All of these flexible ligands displayed
structure-based inhibition on [125I]IMPY (Figure S1 and S2) and the Ki values are listed in Table 1.
Table 1. Inhibition constants for the binding of [125I]IMPY to Aβ1-42 aggregates a
compound
22
Ki
compound
83
Ki
compound
101
Ki
31.0 ± 8.2
15.0 ± 3.6
8.1 ± 0.7
29.6 ± 4.1
>1000
199.6 ± 28.7
258.3 ± 18.3
>1000
23
84
102
24
85
459.9 ± 56.9
24.3 ± 1.0
66.1 ± 7.5
33.6 ± 0.8
12.2 ± 2.7
12.7 ± 3.6
80.5 ± 19.1
26.0 ± 5.0
6.6 ± 1.8
105
25
9.0 ± 1.8
86
106
>1000
26
7.8 ± 3.5
87
115
39.5 ± 13.5
57.2 ± 15.6
74.3 ± 15.9
338.8 ± 160.2
34.3 ± 22.1
632.6 ± 81.9
>1000
27
55.3 ± 16.1
9.3 ± 4.0
88
116
41
89
120
42
36.1 ± 8.4
11.4 ± 1.8
13.7 ± 8.3
6.3 ± 3.9
90
126
43
91
127
44
92
129
45
93
141
46
17.9 ± 4.4
11.6 ± 0.5
446.0 ± 88.8
23.6 ± 1.1
52.1 ± 5.5
10.8 ± 3.8
59.3 ± 17.1
94
3.5 ± 0.9
142
>1000
77
95
4.5 ± 2.5
145
>1000
78
96
4.8 ± 0.7
146
>1000
79
97
501.9 ± 27.0
161.8 ± 54.7
35.6 ± 7.7
216.5 ± 2.7
153
125.6 ± 67.3
26.4 ± 4.3
9.2 ± 3.6
17.3 ± 3.8
80
98
154
81
99
155
82
100
IMPY
a Measured in triplicate with values given as the mean ± SD.
18
ACS Paragon Plus Environment

Page 19 of 43
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
The length effect of different alkane linkage between two phenyl rings were initially explored, as
shown in Figure 3A, when n ≤ 6 (77, 86, 88, 89, 90 and 99), good binding affinity was maintained
(Ki < 50 nM), however, when n > 6 (100, 101 and 102), the binding affinity displayed some
reduction, with Ki values around 200 nM. These results suggest that the flexible diphenoxy
derivatives with a certain length of alkane linkage possess good affinity for Aβ1-42 aggregates, which
is different from the common cognition of the rigid, planar, and π-conjugated systems for Aβ probes.
Then the substitution effect on the phenyl ring were investigated, as shown in Figure 3B and 3C,
by keeping the certain length of alkane linkage (n = 1 and 5), specific para substitution (p-NMe2 and
p-I) on one phenyl ring, and introducing different para substituents (F, Cl, Br, I, NMe2, OCH3 and
t-Bu3) on another ring. First of all, these ligands maintained good affinity for Aβ1-42 aggregates, with
Ki values below 60 nM and most being less than 20 nM, which is comparable or superior to IMPY in
the same condition. Secondly, when para-NMe2 is located on one phenyl ring (Figure 3B), the
ligands bearing long alkane linkage (n = 5) showed better affinity than those bearing short alkane
linkage (n = 1) (Ki values, 80 > 93, 81 > 94, 82 > 95 and 83 > 96), except the para iodinated ligands
(77 < 90); however, when para substitution is iodine atom on one phenyl ring (Figure 3C), the length
of linkage had little impact on affinity (22 vs 41, 23 vs 42, 24 vs 43, 25 vs 44, 26 vs 45 and 27 vs 46).
Thirdly, for p-NMe2 or p-I substituted ligands bearing different lengths of alkane linkage (n = 1 or 5),
changing para substituents on another phenyl ring, the affinity did not have remarkable change with
halogens or electron-donating groups. These results suggest that the para substitution of phenyl ring
has high tolerance for halogens and electron-donating groups.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Having identified optimal substituents (p-NMe2 and p-I) for the phenyl ring that maintains good
19
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 20 of 43
1
2
3
4
5
6
7
8
9
affinity, we then embarked on exploring how the substituents on different positions of another
phenyl ring affect the affinity. As shown in Figure 3D, a common tendency was observed that the
binding affinity decreased dramatically ranging from para, meta to ortho-position, respectively, for
p-NMe2 ligands (n = 1 or 5), 77 (p-I) < 79 (m-I) < 78 (o-I), 90 (p-I) < 92 (m-I) < 91 (o-I), for p-I
ligands (n = 1 or 5), 77 (p-NMe2) < 85 (m-NMe2) < 84 (o-NHMe), 90 (p-NMe2) < 98 (m-NMe2) < 97
(o-NMe2). These results indicate that the position of substituents is important for binding affinity.
This phenomenon has also been observed for dibenzylideneacetones40 and flexible
benzyloxybenzene37 derivatives.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Inspired by the results that flexible structures still maintain good affinity, we attempted to probe
these diphenoxy derivatives bearing various types of more flexible linkages. First, short PEG linkage
(n = 1 - 3), commonly used to adjust the lipophilicity of medicinal agents, was introduced between
the two phenyl rings. As displayed in Figure 3E, these ligands with PEG linkage still retained good
affinity (Ki < 60 nM), which gradually decreased by increasing the PEG unit (n = 1, 2, 3; 77 < 115 <
116). In addition, this tendency was similar to the ligands with different lengths of alkane linkage,
but ligand 116 displayed better affinity than ligand 100 with the same linkage atoms, which means
that adding hydrophilic group (oxygen atoms) in the linkage is favorable for binding. Second, a
piperazine ring, which is a nonlinear chain linkage with two hydrophilic nitrogen atoms, was
incorporated between the two phenyl rings (Figure 3F). The compound 129 was initially evaluated,
which could bind to Aβ1-42 aggregates, although the affinity is weak (Ki = 632.6 ± 81.9 nM). To
reduce the steric hindrance of piperazine ring, the distance from piperazine to phenyl rings was
lengthened by adding certain length of straight alkane chain (n = 2, 4 and 6). These more flexible and
non-planar ligands still kept binding affinity for Aβ1-42 aggregates; moreover, the affinity increased
20
ACS Paragon Plus Environment

Page 21 of 43
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
regularly along with increasing alkane chain (153 > 154 > 155), especially for ligand 155 (n = 6),
which showed excellent binding affinity (Ki = 9.2 ± 3.6 nM), higher than IMPY (Ki = 17.3 ± 3.8 nM)
under the same assay conditions. Third, to test the influence of the alkane linkage (n = 3), we put a
hydroxyl group or a fluorine atom in the middle of an n-propyl linkage, which generates a chiral for
the asymmetric ligands. In a previous study, we reported two pairs of enantiomers with hydroxyl
group, and they exhibited high affinity for Aβ1-42 aggregates.41 In this study, to decrease the
complexity, symmetric ligands with bis-dimethylamino or bis-monomethylamino groups were
selected for discussion. Compared with ligand 87, the introduction of the hydroxyl group barely
affected the binding affinity (87 ≈ 120, with Ki values around 70 nM). Moreover, fluorinated ligand
127 (Ki = 34.3 ± 22.1 nM) with bis-dimethylamino groups displayed better affinity than hydroxyl
ligand 120 and the fluorinated bis-monomethylamino ligand 126 (Ki = 338.8 ± 160.2 nM).
To further investigate the possible SAR of these ligands for Aβ aggregates, one of the phenyl ring in
the diphenoxy ligand was removed (105 and 106) or changed to non-aromatic piperazine (141 and
142) or piperidine (145 and 146) rings. As shown in Table 1, the affinity for these mono-phenyl
ligands to Aβ1-42 aggregates was completely dismissed (Ki > 1000 nM), which suggests that the two
phenyl rings in the flexible structure play a critical role for binding.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Radiochemistry. Iodinated ligands 77, 90, and 115 displayed high affinity for Aβ1-42 aggregates, and
thus they were selected for radiolabelling. Three tributyltin precursors (103, 104, and 117) were
prepared from the corresponding iodinated ligands (77, 90, and 115) according to the methods in
Scheme 1 and 3. Iododestannylation reactions were then applied to generate the radioiodinated
ligands ([125I]77, [125I]90, and [125I]115) with high radiochemical yields of 86.2%, 94.9%, and 92.9%,
21
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 22 of 43
1
2
3
4
5
6
7
8
9
respectively (Scheme 6). The final radiotracers were purified by radio-HPLC with radiochemical
purity greater than 98%. The chemical identities of the 125I-labeled ligands were confirmed by
comparing with the retention times of corresponding non-radioactive iodinated ligands in
co-injection and co-elution on HPLC, and the retention times of each pair matched well, differed by
less than 0.8 min (Table S2).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
22
ACS Paragon Plus Environment

Page 23 of 43
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
Figure 3. Comparison charts of inhibition constants to explore the effect of different linkages and
substituents. (A) Binding affinity of ligands with different length of alkane linker; (B, C) Binding
affinity of ligands with different para substituents; (D) Binding affinity of ligands with certain
substituents in different position; (E) Binding affinity of ligands with different length of PEG linker;
(F) Binding affinity of ligands with hydrophilic piperazine linker.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 6. Synthesis of radioiodinated ligands. a
125I
SnBu3
O
O
O
a
O
O
n
n
[
[
125I]77
125I]90
N
N
N
n = 1
n = 5
103
104
n = 1
n = 5
125I
SnBu3
O
a
2O
2O
N
[
125I]115
117
a Reagents and conditions: (a) 1. [125I]NaI, HCl (1 M), H2O2 (3%); 2. NaHCO3.
Biological evaluations. In vitro digital autoradiography was performed with 125I-labeled diphenoxy
ligands on brain sections of AD patients, transgenic (Tg) mice and control mice to demonstrate high
and specific binding. Consistent with Thioflavin-S fluorescent staining, three 125I-labeled ligands,
[125I]77, [125I]90 and [125I]115, all exhibited excellent matching and distinctive Aβ plaques labeling
without background non-specific labeling on the brain section of AD patients and Tg mice, whereas
Aβ plaques labeling was absent in brain sections of control mice (Figure 4). The brain uptake and
washout properties of these 125I-labeled ligands were evaluated by ex vivo biodistribution studies
using normal ICR mice. As shown in Figure 5 and Table S3, [125I]77 possessed highest initial brain
uptake (6.03 ± 0.77 %ID/g) and fastest washout with brain2min/brain60 min ratio of 7.0, [125I]115 had
moderate initial brain uptake (3.12 ± 0.22 %ID/g) and relatively faster washout (brain2min/brain60 min
= 5.2), whereas [125I]90 displayed poor in vivo brain kinetics with low initial brain uptake (1.41 ±
23
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 24 of 43
1
2
3
4
5
0.08 %ID/g) and slow washout (brain2min/brain60 min = 2.2). Moreover, the radioactivity in mice
6
7
thyroids remained a low level. However, the uptake of [125I]77 and [125I]115 in blood increased over
8
9
time.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. In vitro autoradiography results of [125I]77 (A, B and C), [125I]90 (D, E and F) and
[125I]115 (G, H and I) in brain sections of AD patient (A and G: 91 years old, female, temporal lobe;
D: 64 years old, female, temporal lobe) and mouse brain sections (B, E and H: Tg, C57BL6,
APPswe/PSEN1, 11 months old; C, F and I: wild-type, C57BL6, 11 months old). The presence and
distribution of Aβ plaques in the same sections were confirmed with Thioflavin-S.
24
ACS Paragon Plus Environment

Page 25 of 43
Molecular Pharmaceutics
1
2
3
Figure 5. Comparison of brain uptake (A) and thyroid uptake (B) of [125I]77, [125I]90 and [125I]115 in
4
5
6
normal ICR mice.
7
8
9
DISCUSSION
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In this study, a library of diphenoxy derivatives with different type of more flexible linkers was
constructed and discovered to have good affinity for Aβ aggregates. SAR analysis revealed that the
two phenyl rings are required for Aβ binding, the para- position is well tolerant for halogens and
steric bulk substituents, but the introduction of meta- and ortho- substituents reduced binding affinity.
More importantly, modification on the linkers between the two phenyl rings had great flexibility, and
the affinity was maintained even with a n-heptane or nonlinear chain linker, which challenged the
long-held belief that rigid and planar structure are exclusively favored for Aβ binding. In competition
binding assay, some flexible diphenoxy ligands could efficiently inhibit the binding of [125I]IMPY to
Aβ1-42 aggregates, which indicates that IMPY and flexible diphenoxy ligands share the same binding
sites on Aβ aggregates. However, considering the more flexible structure, quite different from IMPY,
we speculated that there should be a big difference in the binding mode between them.
Global analysis of all these flexible ligands allows us to set up a predicted binding model for
these ligands interacting with Aβ peptide. According to the previous report, there are three binding
sites on Aβ peptide, including two high capacity sites, binding site 1 (BS1) and BS2, which are
adjacent or partially overlapping, and a low capacity site, BS3. Th-T and its analogs, including
IMPY, not only bound with very high affinity to BS3, but also bound with high affinity to either one
of two additional binding sites on the Aβ polymer38. In this study, as described above, we inferred
the binding model of IMPY as shown in Figure 6A. Then, considering that these flexible diphenoxy
derivatives compete with [125I]IMPY, it is reasonable to predict the binding model of diphenoxy
25
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 26 of 43
1
2
3
4
5
6
7
8
9
compounds with different lengths of alkane linkage. As shown in Figure 6B, 6C and 6D, when the
alkane linker is short (n ≤ 3), the whole ligand is located in anyone of BS1, BS2 and BS3, the same
as IMPY; when the alkane linkage is long (n = 4 - 6), the two phenyl rings fail to jam in one binding
site and extend to target two nearby binding sites, like BS1 and BS2; if the alkane linkage is too long
(n = 7, 9 and 11), the two phenyl rings faintly interact with two binding sites. Due to the tension of
long flexible alkane linkage and the force outside, the binding affinity was predicted to decrease by
lengthening the alkane linkage, which was consistent with the experiment results (Figure 3A). In
addition, the effect of substituent position on affinity was relatively obvious for short alkane linkage
(n = 1) compared to long linkage (n = 5) (Figure 3B and 3D), which implied that in one binding site,
the hindering effect between meta- or ortho- substituents and amino acid residues was stronger than
the ligands interacted with two binding sites (Figure 6E and 6F). Similarly, ligands with short PEG
linkage (n = 2 and 3) were could be viewed as occupying two binding sites (Figure 6G), and adding a
hydrophilic group in the linkage is favorable for binding. We speculate that the linkage outside of the
hydrophobic pocket may interact with a hydrophilic environment. Some ligands with hydrophilic
piperazine-modified linkage also displayed good affinity, which supports the above concept (Figure
6H). For this series of ligands, short linkage results in poor affinity due to the locked steric hindrance
of piperazine ring, and this effect could be decreased by increasing alkane linkage. At last, it’s easy
to understand that the monophenoxy derivatives displayed weak affinity due to one binding site
interaction with rotatable linkages outside (Figure 6I). Though the maximum length of flexible
alkane and hydrophilic linkage for this diphenoxy scaffold was not defined in this study, we think
that too long a linkage is unfavorable for the binding because of excessive flexibility of rotation. In
summary, the hypothetical binding model could explain the experimental affinity for these flexible
26
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 27 of 43
Molecular Pharmaceutics
1
2
3
4
5
6
7
diphenoxy ligands, though it is based on limited experimental data in the present study and needs
testing through more experimental data and theoretical calculations.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. The predicted binding model for these flexible ligands on Aβ aggregates. (A) IMPY bind
to one site on Aβ aggregates; (B, C, D) Ligands with different length of alkane linker bind to Aβ
aggregates; (E, F) The effect of substituent position to affinity with short (n = 2) and long (n = 6)
linker; (G) Ligands with different length of PEG linker; (H) Ligands with hydrophilic piperazine ring
binds to Aβ aggregates; (I) Mono-phenoxy ligands fail to bind to Aβ aggregates.
Besides the SAR analysis, compound 77, 90, and 115 were translated to the 125I-labeled versions, and
their biologic properties were further evaluated. Autoradiographic results confirmed the specific
binding affinity of them for native Aβ plaques. Biodistribution studies have shown their blood-brain
27
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 28 of 43
1
2
3
4
5
6
7
8
9
barrier (BBB) penetration and washout properties. The molecular weight and lipophilicity of
radiotracers are two important factors for BBB penetration and washout properties. [125I]77 not only
has a low molecular weight (383 daltons) but also has relatively moderate lipophilicity (Log D =
2.96 ± 0.13), which lead to good in vivo properties. Although [125I]115 possesses considerable
lipophilicity (Log D = 2.95 ± 0.08), it has a higher molecular weight (427 daltons), which resulted in
the lower initial brain uptake. [125I]90 with a high molecular weight (439 daltons) and high
lipophilicity (Log D = 4.15 ± 0.08), showed the lowest initial brain uptake. Compared to the
radioiodinated flexible benzyloxybenzene ligand [125I]BOB-4 (brain2min = 6.18 %ID/g and
brain2min/brain60 min = 16.3) and rigid ligand [125I]IMPY (brain2min = 5.05 %ID/g and brain2min/brain60
min = 11.2)37, [125I]77 has equivalent or better ability to penetrate BBB but slower clearance from the
normal mice brain. Though [125I]77 possesses good BBB penetration and favorable stability, the high
uptake and slow washout in the blood are innegligible issues. Therefore, further structural
optimization of these probes is required to improve their properties in vivo.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In conclusion, these diphenoxy derivatives with flexible linkers have good affinity for Aβ
aggregates, SAR analysis and predicted binding model might give a new sight of rationally designing
of novel Aβ probes, provide a totally new perspective for understanding the binding mode between
probes and Aβ protein, and even offer unexpected guidance for the development of novel
therapeutics against AD or other disease associated with Aβ protein. In addition, radio-iodine labeled
ligands displayed promising properties in vitro and in vivo, which could potentially convert to apply
for the detection of Aβ plaques as SPECT imaging agents. Further studies of theoretical calculation
for clarifying the binding mode and optimization structure modifications for developing potential Aβ
imaging agents are currently underway and will be reported in time.
28
ACS Paragon Plus Environment

Page 29 of 43
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
ASSOCIATED CONTENT
Supporting Information
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The chemical yields, NMR and MS data for all compounds. Additional figures, tables, HPLC files
and NMR/MS spectra. The Supporting Information is available free of charge on the ACS
Publications website at DOI: …..
AUTHOR INFORMATION
Corresponding Authors
* E-mail: cmc@bnu.edu.cn. Phone: +86-13811995064.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (Grant No.
U1967221, 21571022 and 81701757).
ABBREVIATIONS USED
AD, Alzheimer's disease; Aβ, β-amyloid; PET, positron emission tomography; SPECT, single
photon emission computed tomography; CR, Congo red; Th-T, Thioflavin-T; X-34,
1,4-bis(3-carboxy-4-hydroxyphe-nylethenyl)-benzene;
Me-X04,
IMSB,
1,4-bis(4′-hydroxystyryl)-2-methoxybenzene;
(E,E)-1-iodo-2,5-bis(3-hydroxycarbonyl-4-methoxy)styrylbenzene; U.S. FDA, United States Food
29
ACS Paragon Plus Environment